Trypanosoma cruzi is a flagellate protozoan that causes a tropical parasitic disease called Chagas disease. In the course of Chagas disease the infected patient typically passes an acute phase followed by a chronic phase with varying symptoms ranging between mild and life-threatening. Only in the initial phase the patient is responsive to antiparasitic treatment. Some patients develop cardiac or intestinal complications leading to death.
T. cruzi is commonly transmitted to humans and other mammals by an insect vector, the blood-sucking “kissing bugs” of the subfamily Triatominae (family Reduviidae; in German: “Raubwanzen”). The disease may also be spread through blood transfusion and organ transplantation, ingestion of food contaminated with parasites, and from a mother to her fetus. To prevent further spread of Chagas disease it is important to screen blood donations and medical products based on blood for the presence of the parasite and for antibodies against T. cruzi, in particular in mainly infected territories like Mexico, Central and South America and in the Southern United States.
Currently, several serologic diagnostic methods are available to detect infections with T. cruzi, e.g. detection of antibodies against T. cruzi by indirect immunofluorescence, indirect hemagglutination, complement fixation, immunoblot techniques and ELISAs. Also methods of molecular biology (e.g. PCR) and elaborate xenodiagnostic methods are applied. In xenodiagnostics a vector-transmitted infection a laboratory-reared, pathogen-free insect (here: the kissing bug) is allowed to suck blood from a patient. The intestinal contents of the insect are then examined for the presence of the pathogen (here: Trypanosoma cruzi).
Each of these methods shows its own weaknesses and strengths with regard to sensitivity and specificity and accordingly there is no gold standard method available so far.
In the beginning of Chagas assay development for detection of antibodies native antigen lysates were applied and are still being used. However, using lysates only one of the three development stages of T. cruzi is represented in this antigen composition so that there is a certain likelihood to miss infections of the two other stages. More modern assays apply mixtures of recombinant antigens, representing all stages of T. cruzi infection.
Serological assays for detecting antibodies against Trypanosoma cruzi antigens have been widely described in prior art literature. For example Silveira et al. (Trends in Parasitology 2001, Vol. 17 No. 6, p. 286-291) describes T. cruzi recombinant antigens relevant for serodiagnosis have been isolated by several laboratories. Several of these genes have tandemly repeated sequences resulting in proteins showing repeated amino acid sequence motifs.
One of the antigens that is widely used in serological assays for detection of antibodies against T. cruzi is JL7, UniProt database entry no. Q4CS87, also described by the synonyms FRA, Agl and H49 (for review see e.g. Cotrim et al. 1995, Mol Biochem Parasitol 71, 89-98 and Umezawa et al. 1999, J Clin Microbiol, 1554-1560). Also Silveira et al. (supra) describe FRA (JL7) in several mixture of various additional T. cruzi antigens for use in ELISA assays. No specific JL7-related polypeptides are disclosed.
Valiente-Gabioud et al. (Exp. Parasitol. 2011, 127, p. 672-679) discuss the effect of repetitiveness on the immunogenicity and antigenicity of T. cruzi FRA (JL7) protein. It is recommended to use four repetitive repeats to get satisfying ELISA signals from human infected sera. The problem of assay specificity is not addressed and no specific JL7 polypeptides are disclosed.
All antigen variants based on the above mentioned JL7 sequence harbor repeats of 68 amino acids that are highly conserved between strains and isolates of T. cruzi. JL7 is a putative calpain cysteine peptidase of 226 amino acids comprising about 3.5 repeats of said motif of 68 amino acids (see FIG. 1). However, antigens harboring several identical or very similar repeats are often prone to interferences caused by non-specific binding of IgM or IgG molecules or rheumatic factors present in a sample to be analyzed for the presence of anti-T. cruzi antibodies.
The problem therefore can be seen in providing a recombinant JL7 antigen composition and diagnostic method for detecting infections with Trypanosoma cruzi that overcome the disadvantages with respect to interference susceptibility.
The problem is solved by the current invention as specified in the claims. The claimed antigens and compositions as well as diagnostic methods provide an immunodiagnostic solution with high specificity and sensitivity and reliable reproducibility.